Mammals have an internal molecular clock that coordinates physiology into rhythms that coincide with the external solar day, providing enhanced evolutionary fitness by timing the peak activity of integrated biochemical processes. Loss of this internal 24-hour circadian clock leads to diabetes, metabolic syndrome, cancer, and premature aging by disrupting the temporal coordination of physiology with our behavior and the external environment. The long-term goal is to develop a deeper mechanistic understanding of how the molecular clock generates 24-hour timing in humans, in order to capitalize on this temporal regulation to develop new and innovative strategies to treat a broad spectrum of human diseases. The objective in this proposal is to identify the structural basis for transcriptional regulation by the primary circadian transcription factor, CLOCK:BMAL1, which controls expression of nearly 15% of the genome on a daily basis to drive circadian rhythms of physiology. Despite its critical importance in human physiology, very little is known about what governs the temporal switch from active to repressive CLOCK:BMAL1 complexes that create the intrinsic 24-hour period of the molecular clock. The central hypothesis is that CLOCK and BMAL1 transcriptional activation domains use intrinsic flexibility to regulate binding of activator and repressors to contribute to 24-hour timing of the molecular clock. Using nuclear magnetic resonance spectroscopy, quantitative biochemistry and cell-based studies, we will pursue two specific aims investigating 1) how a dynamic conformational switch in the BMAL1 activation domain regulates CLOCK:BMAL1 activity and 2) how competition for binding to the CLOCK activation domain regulates CLOCK:BMAL1 in normal clock function and in human cancer. Our innovative approach integrates diverse techniques from cell biology to solution NMR spectroscopy to generate biomedically relevant, atomic-level insight into clock function. The proposed research is significant, because it is expected to provide fundamentally new conceptual advances that address how CLOCK:BMAL1 works to generate 24-hour molecular rhythms and control global homeostasis. Ultimately, such knowledge has the potential to inform new strategies for basic and translational research into circadian control of human physiology.